Adenovirus E1-complementing cell lines

ABSTRACT

The present invention relates to adenovirus (Ad) E1-complementing cell lines which significantly reduce the presence of replication competent Ad (RCA) and can serve for the large scale production of infectious E1-deleted adenoviral particles that may be used for the treatment human patients as for example in gene therapy. As well the invention relates to a method for the large scale production of recombinant infectious adenoviral particles harboring an exogenous sequence of interest and to a RCA-free stock of infectious adenoviral particles. The invention further relates to a recombinant vector for transfecting an eukaryotic cell line in order to construct Ad E1-complementing cell lines which significantly reduce the presence of RCA and to a method therefor.

FIELD OF THE INVENTION

The present invention relates to the introduction of exogenous geneticsequences into cells, cell lines or organisms, and to gene transfer andgene therapy. The invention further relates to defective adenoviralvectors and E1-complementing cell lines. More specifically the presentinvention relates to adenovirus (Ad) E1-complementing cell lines whichsignificantly reduce the presence of replication competent Ad (RCA) andcan serve for the large scale production of E1-deleted Ad vectors thatmay be used for the treatment of human patients. As well the inventionrelates to a method for the large scale production of recombinant Adharboring an exogenous sequence of interest.

BACKGROUND OF THE INVENTION

Therapeutic strategies in various discases include: nonspecific measuresto mitigate or eliminate a cell dysfunction and prevent cell death;replacement of a missing or malfunctioning protein; introduction offunctional nucleic acids (RNA or DNA) into cells to replace a mutatedgene and introduction of novel genetic constructs to alter a cellularfunction. Advances in DNA technology have had a major impact on each ofthose therapeutic possibilities and nucleic acid transfer into diseasedcells appears by far the most promising modality (Mulligan 1993, Science260:926-932).

Viral vectors permit the expression of exogenous genes in eukaryoticcells, and thereby enable the production of proteins which requirepost-translational modifications unique to animal cells.

The wealth of information accumulated on adenoviruses over the lastdecades, has promoted them at the forefront of the gene therapy orimmunization fields. Several features of adonoviruses make themattractive as gene transfer tools: (1) the structure of the adenoviralgenome is well characterized; (2) large portions of viral DNA can besubstituted by foreign sequences; (3) the recombinant variants arerelatively stable, (4) the recombinant virus can be grown at high titer;(5) no human malignancy is associated with adenovirus; and (6) the useof attenuated wild-type adenovirus as a vaccine is safe.

Ad ate thus considered as very good vector candidates for in vivo genetransfer. Generally, such vectors are constructed by inserting the geneof interest in place of essential viral sequences such as E1 sequences(Berkner 1988 BioTechniques 6:616-629; Graham et al., 1991, Methods inMolecular Biology, 7:109-128, Ed: Murcy, The Human Press Inc.). Thisinsertion results in an inactivation of the Ad since it can no longerreplicate, hence the term replication-defective Ad. In order topropagates such vectors must be provided with the deleted element, (i.e.E1 proteins).

The elucidation of the nucleotide sequence of many Ad subtypes hasenabled a precise characterization of the genomic organization thereof.The nucleotide sequence of human Ad5 is available from GenBank underaccession number M73260. In simplistic terms adenoviruses comprise: (1)two inverted terminal repeats (ITR) at each end (5' and 3') which areessential for viral replication; (2) the early region 1 (E1) containingthe E1A and E1B regions, both indispensible for replication, E1A and E1Bare also required for complete transformation of various rodent celllines, and polypeptide IX (pIX) which is essential For packaging offull-length viral DNA; and (3) the E2, E3 and E4 regions, with E3 beingdispensable for replication (reviewed in Acsadi et al., 1995, J. Mol.Med. 73:165-180).

Recently, human Ad serotypes 2 and 5 have been used as vectors forefficient introduction of genes into several cell types both in vitroand in vivo (reviewed in Trapnell et al., 1994, Current Opinion Biotech.5: 617-625; and Acsadi et al., 1995, J. Mol. Med. 73:165-180). Severalfactors need to be taken into consideration during the generation of Adrecombinants, among them is the impaired growth characteristics of someof them ( Imler et al., 1995 Gene Ther. 2:263-268; Massie et al., 1995Bio/Technol. 13:602-608; and Schaack et al., 1995 J. Virol. 69:3920-3923) which complicate the screening, propagation and production ofhigh quality recombinant viral stocks with high titers (more than 10¹¹pfu/ml). Recently, critical issues relating to the characterization ofsuch Ad vectors for gene therapy were reviewed in relation to clinicaltrials of the cystic fibrosis gene therapy (Engelhardt et al., 1993Nature Genetics 4:27-34; Zabner et al., 1993 Cell 75:207-216; Boucher etal., 1994 Human Gene Ther. 5:615-639; Mittereder et al., 1994 Human geneTher. 5:717-729; and Wilmot et al., 1996 Human Gene Ther. 7:301-318).Presently, a number of human clinical trials making use of Adrecombinants for the treatment of diseases like cystic fibrosis,Duchenne muscular dystrophy, and cancer, have started or are beingconsidered (Lochmuller et al., 1994 Hum. Gene Ther. 5: 1485-1491).Potential sites for the insertion of a gene of interest in therecombinant Ad vectors comprise the E1 or E3 regions (i.e. E1+E3-deletedAd recombinants) or the region between the end of the E4 and thebeginning of the 3' ITR sequences. The majority of in vivo gene transferexperiments and human trials have been carried out using E1- andE3-deleted human type 2 or 5 adenoviruses. As alluded to above,E3-deleted recombinants are replication competent E1-deletedrecombinants however, are unable to replicate and the missing E1 geneproducts are provide in trans by the E1-complementing cell line 293(Lochmuller et al., 1994 Hum. Gene Ther. 5: 1485-1491). The 293 cellswere established by stable transfection of a human embryonic kidney cellwith adenoviral (human type 5) DNA containing the full length E1 region.The maximum deletion of up to 2.9 kb in the E1 region leaves intact theITR sequence, the packaging signal at the left and of the adenoviral DNA(188-358 bp) and the pIX coding region (starting at 3507 bp). A usefulE3 deletion was made by deletion of a 1.9 kb Xba I fragment (79 and 85mu). These combined E1 and E3 deletions allowed for insertingapproximately 7 kb of foreign DNA sequences in this first-generationrecombinant. Extensions of the deletion in the E3 regions furtherincreased the insert capacity to 8 kb, which meets the size requirementsfor most of the gene therapeutics (Bett et al., 1994 Proc. Natl. Acad.Sci. 91:8802-8806).

It is important to note that the recombinant Ad produced for clinicaluse have all been obtained using 293 cells (Graham et al., 1977, J. Gen.Virol. 36:59-72). Until the present invention, 293 cell line was theonly available complementation cell line which efficiently expressed E1Aand E1B RNAs and proteins. Unfortunately, it hats been documented thatreplication competent, also termed "revertant" virus can appear duringmultiple passages of the E1- and E3-deleted recombinant Ad in 293 cells,and eventually outgrow the original recombinant in large scalepreparations (Lochmuller et al., 1994 Hum. Gene Ther. 5: 1485-1491). TheE1 region is acquired from the 293 cells (and its derivatives) byhomologous recombination at a very low frequency, but the E1-positiverevertants seem to have a growth advantage with respect to theirE1-negative counterparts. The presence of these revertants could thusjeopardize the safely of human gene therapy trials, especially when oneconsiders the number of infectious viral particles required in certainapplications. Experiments performed with mouse muscle have taught theuse of of 2×10⁹ virus particulars to transduce more than 80% of themuscle fibers, since a human muscle is 2500 times larger, that wouldtranslate in the use of approximately 10¹² -10¹³ viral particulars toinject only one human muscle. Supposing the presence of as little as1/10 particules of E1+ revertants, in the stock, 10³ -10⁴replication-competent particules would be injected in the muscle. It isclear that such an approach would fail to satisfy regulatory agencies.

Indeed, the 293 cells have been deemed "not suitable for large scaleproduction of clinical grade material since batches are frequentlycontaminated with unacceptably high levels of replication competentadenovirus (RCA) arising through recombination" (Imler et al., 1996 GeneTher. 3: 75-84). It should be stressed that the same authors havereported that numerous attempts to construct stable and efficientE1-complementing cell lines have failed and is therefore not a trivialtask. In an attempt to solve this problem of RCA generation (Imler etal., 1996 Gene Ther. 3: 75-84) produced an E1-complementing cell line bystably transforming human lung A549 cells with E1 sequences containingthe E1A, E1B and pIX regions. Novel A549 E1-complimenting cell lineswere obtained which express high levels of E1 RNA and proteins.Strikingly however, the authors were unable to detect E1B proteinexpression in any of the A549 clones analyzed whether or not theyproduce high level of E1B RNA. Thus, the presence of a functionalgenetic unit does not necessarily predict that upon stable integrationin the host, it will give rise to the expected proteins. It is alsoreported therein that the A549 clones, testing positive for infectionwith E1-deleted Ad vectors, showed a transformed phenotype and that theamplification yields therewith arc significantly lower than thoseobtained with 293 cells. Unfortunately, the generation of RCA with theseA549 cells was not assessed. It should be noted that in the Imler etal., constructs, a significant overlap between the complementing elementand the defective adenoviral vector occurs at the 3' end of the E1region (approximately 700 bp). It follows that this overlapsignificantly increases the probabilities of homologous recombinationand hence of the production of E1+ revertants. A disclosure of defectiveAd vectors for the expression of exogenous nucleotide sequences in ahost cell or organism, as well as vectors for the construction ofE1-complementing cell lines, along the same lines is also found in theFrench publication to Imler et al., WO94/28152. However, this documentfails to give an assessment of the yield of production of recombinant Adby the complementation cell line, of the expression of the differentadenoviral transcripts and proteins by the complementing cell line, andvery importantly of the presence or absence of RCA during the productionprocess leading to the obtention of the stock of defective Ad harboringthe exogenous sequence of interest. It should be noted that WO94/28152claims to diminish the problem of RCA production by deleting the 5' ITR(a non-substantiated declaration).

There still remains a need for the description of an E1-complementingcell line which combines at least one of the following properties: itexpresses E1A and E1B proteins; it minimizes or abrogates the productionof E1+ revertants; it is substantially as efficient as 293 or itsderivatives in producing recombinant Ad; and it does not show atransformed and rounded phenotype. It would thus be advantageous to beprovided with such E1-complementing cell lines which are efficient forthe large scale production of E1-deleted Ad vectors devoid of RCA.

All of the above-cited citations are herein incorporated by reference.

SUMMARY OF THE INVENTION

An object of one aspect of the present invention is therefore to providean E1-complementing cell line which satisfies at least one of thefollowing properties: (1) it expresses functional E1A and E1B proteins;(2) it minimizes the production of E1+ revertants; (3) it issubstantially as efficient as 293 or its derivatives in producinginfectious recombinant Ad; and (4) it does not show a transformed androunded phenotype.

Another object of one aspect of the present invention is to provide amethod for the large scale production of E1-defective recombinant Adwhich minimizes the production of RCA.

An additional object of one aspect of the present invention is toprovide a recombinant adenovirus construct for the establishment of anE1-complementing cell line in accordance with the present invention.

Yet another object or one aspect of the present invention is to providea therapeutic use of an E1-complimenting cell line in accordance withthe present invention.

A further object of one aspect of the present invention is to provide amethod of treatment by which a therapeutically or prophylacticallyefficacious quantity of a recombinant Ad, produced in anE1-complementation cell line in accordance with the present invention,or an E1-complementation cell line in accordance with the presentinvention harboring a recombinant Ad is administered to a patient inneed of such a treatment or prophylaxy.

More specifically, in accordance with the present invention, there isprovided an adenovirus (Ad) E1-complementing cell line having a stablyintegrated complementation element comprising a portion of the AdE1region covering the E1A gene and the E1B gene but lacking the 5'inverted terminal repeat (ITR), the packaging sequence, and the E1Apromoter; the E1A gene being under control of a first promoter elementand the E1B gene being under control of a second promoter element, thestably integrated complementation element giving rise to functional E1Aand functional E1B proteins, whereby the stably integratedcomplementation element complements in trans a defective adenoviralvector and does not generate replication competent adenovirus (RCA)produced by homologous recombination between said defective adenoviralvector and said complementing element at a detectable level.

In accordance with the invention, there is also provided a method forlarge scale production of infectious E1-defective adenoviral particlescomprising: a) transfecting an E1-defective adenoviral vector into anE1-complementing cell line to obtain plaques; b) screening the plaquesto identify plaques positive for E1-defective adenovirus (Ad); c)submitting the E1-defective Ad of b) to at least two rounds of plaquepurification by infection into an E1-complementing cell line to obtainsubstantially pure infectious E1-detective adenoviral particles, and d)scaling up production of the substantially pure infectious E1-detectiveparticles of c) by infecting an E1-complementing cell line and growingthe cell line to obtain a concentrated stock of infectious E1-defectiveadenoviral particles, wherein in at least one of steps a), b), c), andd), the E1-complementing cell line of claim 1 is used, therebyminimizing the production of RCA in the concentrated stock ofE1-defective Ad infectious particles obtained in d). In addition thereis provided a RCA-free stock of defective adenoviral vector produced inaccordance with the above-recited method.

In accordance with the present invention, there is also provided arecombinant vector for constructing an Ad E1-complementing cell line inaccordance with the present invention and a method of producing an AdE1-complementing cell line comprising: a) transfecting a euaryotic cellline with a recombinant vector according to the present invention; b)selecting a cell having stably integrated the complementation element;and c) selecting the cell of b) expressing functional E1A and E1Bprotein selecting the cell of b) expressing functional E1A and E1Bproteins and complementing an E1-defective adenoviral vector so as toyield of infectious E1-defective adenovirus particles while avoiding thegeneration of a detectable level of RCA.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally describe the invention, reference will now be madeto the accompanying drawings, showing by way of illustration a preferredembodiment thereof, and in which:

FIG. 1 shows the A 549-AdE1-complementing cell lines; in A is shown aschematic representation of pHβE1AE1B which was used to construct theA549 E1-complementing cell lines, as well as a comparison of the overlapbetween the sequence thereof and that of the defective adenovirus (Ad5ΔE1ΔE3); in B is shown immunoblots of A 549 cell lines probed withantibodies to E1A (45K) and E1B (19 kDa and 55 kDa) proteins; lanes 1,293 control lanes 2, A549 control; lanes 3, BMAdE1-78; and lanes 4BMAdE1-220; and

FIG. 2 shows a diagram depicting viral multiplication of Ad5CMVlacZ inBMAdE1 clones compared to 293 cells.

Other objects, advantages and features of the present invention willbecome more apparent upon reading of the following non restrictivedescription of preferred embodiments with reference to the accompanyingdrawings which are exemplary and should not be interpreted as limitingthe scope of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to E1-complementing cell lines which cancomplement E1-defective adenoviral vectors while significantly reducingthe presence of E1+ revert ants which have become replication competent(RCA) through recombination with the adenoviral sequences present in theE1-complementing cell line. The minimizations if not the totalabrogation of RCA, is crucial if the adenoviral vector harboring anexogenous genetic sequence is to be used in human therapies such as genetherapy. It should be understood that PCR analysis is not sensitiveenough to assess the level of purity of an infectious viral stockwarranted by the regulatory agencies. The detection of RCAs is thusgenerally based on tedious plaque assays. The limit of detection of RCAcurrently available to the skilled artisan is approximately between oneRCA per 10⁷ to 10⁸ (perhaps 10⁹) infectious particles. Preferably, thenumber of RCA in the final adenoviral infectious stock should beinferior or equal to approximately one RCA per 10⁹ infectious particleand especially preferably inferior or equal to approximately one RCA per10¹⁰ infectious particles.

According to the present invention, when relating to adenoviral vectors,adenoviral sequences, it should be understood that they can be derivedfrom a natural or wild type adenovirus or preferably from a canine,avian or human adenovirus, more preferably a human adenovirus of type 2,3, 4, 5 or 7 and especially preferably a human adenovirus type 5 (Ad5).In the preferred embodiments described herein, Ad5 was used and thenucleotide positions referred to are taken from the nuclcotide positions532-3525 as described in GenBank under the reference N° M73260.

It should also be understood that numerous E1-defective adenoviralvectors are encompassed by the scope of the present invention. One ofthe crux of the invention lying in a minimization of the formation ofRCA, a particular E1-defective adenoviral vector should be chosen so asto minimize the presence of homologous sequences between the defectiveadenoviral vector and the complementing element stably integrated intothe genome of the E1-complementing cell line chosen. It will beunderstood that the complementing cell line should provide the essentialelements lacking in the Ad-defective vector used. Defective adenoviralvectors contemplated within the scope of the present invention includewithout being limited thereto, vectors which comprise in the 5' to 3'direction, the 5' ITR, the packaging sequence, the E2 region, the E4region the 3' ITR as well as the Major Late transcript region. Since theE3 region is dispensable for replication, that region can be deletedfrom the Ad vector thereby permitting the insertion of a largerexogenous genetic sequence therein. In a preferred embodiment, thedefective adenoviral vector used is Ad5 ΔE1 ΔE3. It is also contemplatedthat a defective adenoviral vector having additional deletions inessential regions (such as E2 and/or E4) can also be used with the celllines of the present invention, provided that all defective elementsthereof are complemented. For example E2 and/or E4 could be supplied inthe E1-complementing cell lines of the invention by a cotransfection ofthe defective Ad with a vector providing the lacking essentialelement(s), thereby complementing all the replication defects of thechosen defective adenoviral vector. The term viral "particle" is wellknown in the art.

The terms "deletion or deleted" should be understood to mean the removalof at least one nueleotide from the targeted region. As well, thisdeletion can be continued or discontinued. Deletions which remove largeportions of the targeted regions are preferred over small deletions,since they diminish the possibility of homologous recombination betweenthe defective adenoviral vector and the complementation element stablyintegrated in the chromosome of the complementing cell line. Thedeletion can be a partial or total deletion of the targeted region.

The defective adenoviral vectors to be used according to the presentinvention arc incapable of replication but gain the capability ofreplication and encapsidation in the complementing cell line whichsupplies the defective products in trans. This generates an adenoviralparticle which is still defective, since it is incapable of replicatingin an autonomous fashion in a cellular host but is neverthelessinfectious since it can deliver the vector to the host cell it inflects.

The term "exogenous nucleotide sequence" is meant to cover nucleic acidssuch as coding sequences and regulating sequences which are generallynot present in the genome of adenoviral viruses. It is also to beunderstood that the exogenous nucleotide sequences should have necessaryinformation to be expressed inside the host cell towards which thedefective adenoviral vector is ultimately targeted. It is to beunderstood that the exogenous sequence can also be expressed in thecomplementing cell line. The exogenous sequences are introduced in theadenoviral vector by the classical techniques of genetic engineeringbetween the packaging sequence in 5' and the 3' ITR. The exogenousnucleotide sequence can contain one or many genetic sequences ofinterest and preferably the gene(s) of interest have therapeutic orprophylactic potential. Such a gene of interest can code for anantisense RNA or a mRNA which can be translated into a protein ofinterest. The gene of interest can be a genomic copy, a cDNA or achimera of both. It can code for a mature protein, a precursor thereof,a protein chimera, a fusion protein, mutant or modified versions of allsuch proteins. The mutant protein can be obtained by way of mutation,deletion, substitution and/or addition of the nucleotide sequenceencoding the initial protein. The exogenous sequence can be natural,genetically engineered, synthesized chemically or combinations thereof.

The gene of interest can be placed under the control of appropriatecontrol elements ensuring the expression thereof in the host cell. Theappropriate control elements comprise transcription elements, generallyknown as promoter elements and enhancer elements (promoter/enhancerelements), and the translation elements. Herein, the terms promoterelement and promoter/enhancer elements are used interchangeably in thebroad sense as control elements. The promoter/enhancer elementcontrolling the transcription can be either a constitutive or aregulatable or inducible promoter of eukaryotic or viral origin. It canalso be the normal control element for the gene of interest Eukaryoticpromoter/enhancer elements are well known to the skilled artisan and canbe readily inserted by standard genetic engineering practices in frontof the gene of interest or modified to suit the proper need thereof. Thepromoter/enhancer element can also be tissue specific. Thepromoter/enhancer element include but are not limited to the SV40 earlypromoter region, the RSV promoter (in the 3' LTR), the TK HSV promoter,the regulatory sequence of the metallothionine gene, tile insulinecontrol region which is active in pancreatic B cells, immunoglobulingene control region which is active in lymphoid cells, mouse mammarytumor virus control region which is active in testicular, breast,lymphoid and mast cells, and human beta actin promoter. Preferably thepromoter/enhancer element controlling E1A is a strong promoter.

Genes of interest which are encompassed by the scope of the presentinvention are in essence illimited since a skilled artisan can adapt byconventional method the teachings of the present invention to theexpression of his own favorite gene of interest. It should be understoodthat a limiting factor is the packaging limit of the defectiveadenoviral vector. In any event, without being limited thereto gene ofinterest includes: growth factors, receptors, for such growth factors orfor other molecules as well as for pathogens, suicide genes, factorsinvolved in blood coagulation, dystrophin, insulin, genes involved incellular transport such as the cyctic fibrosis transmembrane conductanceregulator, or the natural resistance associated macrophage protein gene,genes coding for antisense or inhibitors of pathogenic organisms,inhibitors of defective metabolic processes or inhibitors of pathogens,cancer suppressor genes, genes expressing transdominant proteins, genesencoding antigenic epitopes or variable regions from specific antibodiesand imnunomodulator genes.

It should be understood that the adenoviral vectors of the presentinvention need not contain only genes or nucleotide sequences havingtherapeutic or prophylactic potential. Nevertheless the non-limitatingapplications of the present invention to gene therapy include atargetting of the following tissues: lung, muscles, liver, kidney,spleen, the nervous system and macrophage. Non-limitative examples ofdiseases for gene therapy include cystic fibrosis, hemophilia andcancer.

The host cell which is to be chosen to eventually become anE1-complementation cell line in accordance with the present inventioncan be chosen among a variety of eukaryotic host cells by a skilledartisan. Advantageously, it will be a mammal cell line or preferably ahuman cell line.

The complenentatioLi cell line according to the present invention can bederived from an immortalized cell line or a primary cell line. Inaccordance with the present invention, one of the crux of which is tominimize the extent of homologous region between the complementationelement and the adenoviral sequences in the defective adenoviral vector,the Eukaryotic E1-complementing cell line according to the invention ortheir derivatives should minimize the formation of E1+ revert ants orRCA. RCA refers to replication competent adenoviruses which are nolonger defective for replication and packaging and can therefore infectcells and lead to toxicity and deleterious immunological reactions.Preferably, the E1-complementing cell lines according to the presentinvention do not yield detectable RCA by PCR analysis and/or plaqueassay on non-complementing cell lines such as A549. More preferably, theE1-complementing cell lines according to the present invention yield anumber of RCA per number of infectious viral particles which isinsufficient to pose a hazard to a patient when a therapeutic orprophylactic dose of adenoviral infectious particles is administeredthereto. Especially preferably, the E1-complementing cell lines of thepresent invention give rise to no RCA.

The cell line to be chosen to become the E1-complementing cell line ofthe invention should be a pharmaceutically acceptable cell line. Theterm pharmaceutically acceptable cell line is meant to refer to the factthat this cell line has been characterized (in terms of history andorigin) and/or has been used for the production of products destined forhuman use (production of material for clinical assays or materialdestined for sale). Such cell lines are available in depositories suchas the ATCC. Without being limited thereto, such cell lines includehuman carcinoma cells A549, human pulmonary cell line MRC5, humanpulmonary cell W138, KB cells, Hela cells and 143 cells. Mostpreferably, the chosen cell line is the A549 cell line. The A549 and 293cell lines for example, grow in monolayer. Other cell lines are able togrow in suspension which permits an easier scaling up of productionsince much larger volumes of cells can be grown. The derivative of the293 E1-complementing cell line, 293S, possesses this advantageousproperty of growing in suspension instead of on a solid support.

The method used to stably integrate the complementation element can beperformed by standard genetic engineering procedures (Graham et al.,1991, Methods in Molecular Biology, 7: 109-128, Ed.: Murey, The HumanPress Inc.) and as described by the present invention. The"complementation element" as used herein refers to a nucleic acidelement which can in trans, complement tie replication defect of thedefective adenoviral vector used. The E1-complementation cell line isthus capable of producing the protein(s) which is necessary for thereplication and packaging of the E1-defective adenoviral vector. Itshould be understood that the complementation element could be mutatedby deletion and/or addition of nucleotides, as long as thesemodifications do not alter the complementation capacity thereof.

In accordance with the present invention, the complementing element, isexpressible, and gives rise to tie E1A and E1B mRNAs as well as to thefunctional E1A proteins (289aa and 243aa) and E1B proteins (19 KDa and55 KDa). In accordance with a preferred embodiment of the presentinvention, the E1A promoter enhancer element has been replaced by thehuman beta-actin promoter, while the E1B enhancer promoter element isthe natural E1B promoter. Of course, these elements can be substitutedby other types of enhancer promoter elements which are well known to theskilled artisan. In addition, they could be mutated or modified so as toadapt the expression of E1A and E1B to a particular situation.Advantageously, the complementing element comprises a transcriptiontermination signal and a polyadenylation signal, and preferably those ofSV40. In a preferred embodiment of the present invention, thecomplementation element comprises the nucleotide sequence betweennucleotides 532 to 3525 of human Ad type 5 as disclosed in Genbank underreference M73260.

The vector of the present invention enabling the construction of theE1-complementing cell line, should comprise the complementing elementwith the necessary control elements, as well as a selection markerpermitting an assessment of the stable integration of thecomplementation element into the genome of the host cell. Such selectionmarkers include but are not limited to neomycin resistance (G418),hygromycin resistance, phleomycin resistance and puromycin resistance.In another embodiment, the selectable markers could be supplied by aco-transfected vector. It is also possible to synthesize by way of PCRor chemically the nucleotide sequences required to construct anE1-complementing cell line in accordance with the invention. Since it isa preferred embodiment to transfect a cell line with a linear fragmentcomprising the E1-complementing element, the E1-complementing element(or cassette) need not necessarily be on a vector. Such a cassette couldbe for example co-transfected with a vector providing a eukaryoticselectable marker which enables stable integration of the complementingcassette in the genome of the transfected cell. This cassette preferablycomprises non-E1 region nucleic acid sequences favoring the integrationof a functional E1-complementing element.

In a preferred embodiment of the present invention, the selectablemarker is SV2-neo. Preferably, the vector used to construct theE1-complementing cell line will also contain a selectable marker and anorigin of replication enabling replication and selection in amicroorganism. Selectable markers and origins of replication formicroorganisms such as bacteria and lower cukaryotes are well known inthe art. The former include without being limited thereto antibioticresistance, auxotrophic markers and the killer gene system.Non-limitative examples of origins of replication include the standardColE1 type for bacteria and the 2μ for yeast.

It is also within the scope of the invention to use the complementingcell line harboring a defective adenoviral vector which comprises anexogenous sequence of interest, directly by implantation into a patient.For such an embodiment an E1-complementing cell line could be derivedfrom cells taken from the patient, transfected with the defectiveadenoviral vector containing the exogenous sequence of interest andimplanted back into the patient. Thus, the present invention alsoencompasses a therapeutic or prophylactic use of a vector containing thecomplementing element, for deriving an E1-complementing cell line, andthe E1-complementing cell lines themselves. In addition,E1-complementing cell lines of the present invention can be used in amethod for the preparation of the infectious adenoviral particles whichcan then be administered to a patient. The administration of suchinfectious particles for a therapy or prophylaxy, are known to thoseskilled in the art, since such technologies have been used in a clinicaltrial for the treatment of cyclic fibrosis for example.

The method of preparation of infectious adenoviral particles accordingto the present invention, is also based on one of the crux of theinvention, the minimization of formation or RCA particles. It will thusbecome apparent, that the cell lines of the present invention offer asignificant advantage over the available complementing cell lines whichgive rise to a significant number of RCA. Since RCA can outgrow theE1-defective adenoviruses, appearance of RCA early in the course ofproduction of the infectious adenoviral particles could negate the usingof a stock of these infectious particles for therapy or prophylaxy. Itis of course known that in human gone therapy trial, safety issues areof paramount importance. One of the key requirements is the stablepurity of the therapeutic Ad recombinant stocks. Thus, the use ofE1-complementing cell lines according to the present invention duringthe course of production of the viral stock, can be of criticalimportance for the obtention of a stock of infectious particles whichcan be administered to patients. It will be understood that the protocolfor the production of these infectious particles can be adapted in avariety of ways, by using for example only E1-complementing cell linesaccording to the present invention, or using other availableE1-complementing cell lines in different phases of the scaling upprocedure as long as the number of passages in a complementing cell linewhich gives rise to RCA is minimized.

The therapeutic maid prophylactic uses which are envisioned as fallingwithin the scope of the present invention are related to the type ofexogenous sequence which is inserted into the defective adenoviralvectors described above. Pharmaceutical compositions in accordance withthe present invention can be manufactured by conventional method. Inparticular, a therapeutically efficacious quantity of a defectiveadenoviral particle produced in accordance with the present invention orof an E1-complementing cell line harboring such an adenoviral particlewill be mixed with a suitable support or carrier. Compositionsencompassed by the present invention can be administered by way ofaerosol or any other conventional fashion known in the art, inparticular by oral, sub-cutaneous, intramuscular, intravenous,intra-peritoneal, intra-pulmonary or intra-tracheal routes. Theadministration can be in unit dose or repetitive doses with varyingintervals in between doses. The administration of the appropriate dosewill vary in accordance with different parameters including theindividual to be treated, the disease, the type of exogenous sequenceharbored by the adenoviral particle and the type of exogenous sequenceharbored by the defective adenoviral particle. As a general rule, thehealth practitioner will adapt the dosage in accordance with those andother parameters.

The emergence of replication-competent E1+ revert ants in stocks ofreplication-defective Ad recombinants (ΔE1+ΔE3) which has beendemonstrated (Lochmuller et al., 1994 Hum. Gene Ther. 5:1485-1491), ismost likely due to a recombinational event, which occurs at very lowfrequency, between the complementing element and the defective Ad.Although the population of replication-competent Ad is found at very lowlevel in early passages, this population dramatically increases duringthe cycles of amplification required to produce large Ad stocks for genetherapy experiments (Lochmuller et al., 1994 Hum. Gene Ther. 5:1485-1491).

The present invention aims at solving this E1+ revertant problem.

MATERIAL AND METHODS

Cells and viruses

293 E1-transformed human embryonic kidney cells (Graham et al., 1991,Methods in Molecular Biology, 7: 109-128, Ed: Murey, The Human PressInc.), A549, and Hela S3 cells were purchased from ATCC and grown at 37°C. in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10%fetal bovine serum (Hyclone) and 2 mM glutamine (Gibco). 293 and BMAdE1clones were infected with AdCMVlacZ (Acsadi et al., 1994 Hum. Mol. Gen.3:579-584) and AdGFP (a recombinant adenovirus expressing greenfluorescent protein) at a MOI of 5-10.

Protein analysis

Cells were harvested, washed in PBS and lysed in Laemmli buffer (10%glycerol, 80 mM tris pH 6.8, 2% SDS). Protein concentration wasdetermined by a Lowry's modified method using the De kit™ (Bio-Rad).Proteins were their separated by SDS-PAGE on a NOVEX™ 10 or 12% precastgel (Helixx).

Western blot hybridization

Proteins were transferred to a Hybond™-C nitrocellulose membrane(Amersham) in a Bio-Rad apparatus. Membranes were blocked overnightusing 5% milk in TBS, and hybridized with the appropriate antibodies.Washes were carried out with 0.1% Tween-20™ in TBS. Revelation was madeby chemiluminescence using the ECL™ kit (Amersham).

In vitro plaque assay with 293 and BMAdE1 cells

Different dilutions of virus (AdCMVIacZ or AdGFP) were plated on 5×10⁵cells in a 60-mm dish and overlayed with 1% sea-plaque™ agarose (FMC).Plaques were observed between 7 to 14 days after infection, overlayedwith Bluo-gal™ (1% sea-plaque™ agarose, 0.3% NP40™, and 0.2% Bluo-gal™from Gibco) if needed, and counted. The plaques were observed using aninverted fluorescence microscope for the AdGFP infections.

Indirect plaque assay by beta-gal expression in Hela S3 cells

Hela S3 were infected with virus stocks obtained by infecting BMAdE1and293 cells with AdCMVlacZ. Different volumes of virus were used in orderto obtain a value in the linear range of the beta-gal assay. A standardcurve was niade with the AdCMVlacZ stock used for tie stock infections.

Beta-gal assay

Beta-galactosidase assays were performed by a chemiluminescent detectiontechnique using the Galacto-Light™ kit (Tropix). Cells were harvested,washed twice with PBS and resuspended in lysis buffer (100 mM KPO₄ pH7.8, 0.2% Triton X-100 ™, 1 mM fresh DTT) at a concentration of 1×10⁶cells in 100 μL. Reaction buffer (70 μL of Galacton™ in 100 mM NaPO₄ pH8.0, 1 mM MgCl₂) was then added to cell extracts (10 μL) in aluminometer cuvette and the cuvette was placed in the luminometer(Berthold) after 1 hour of incubation. 100 μL of Accelerator (10%Emerald enhancer in 0.2M NaOH) were then injected and the sample wascounted for 10 seconds. The positive control consisted of 1 μL ofbeta-galactosidase from Sigma (in 01M NaPO₄ pH 7.0, 1% BSA) added Lo amock cell extract while The negative control was a mock cell extract.

CONSTRUCTION OF pHβE1AEIB

As a first step in the production of E1-complementing cell lines of theinvention, an expression vector containing Ad sequences was constructed.The genetic engineering methods used for the construction of the vectorsand cell lines of the present invention are well known methods in thearts. Restriction endonucleases and other DNA modifying enzymes wereused according to the supplier's recommendations or according tostandard protocols such as that of Sambrook et al., (1989 CSH press; thecontents of which is heroin incorporated by reference). Transformationof bacteria, purification of plasmids, transfection, and other molecularbiology assays were also performed in accordance with known methods suchas found in (Sambrook et al., 1989 CSII press). Escherichia coli DH5strain was made competent and transformed plasmid DNA was prepared bythe alkaline lysis method and purified by CsCl-ethidium bromide densitygradient centrifugation.

Briefly, the plasmid pHβE1AE1B was constructed by subcloning the 3.0 kbgenomic DNA of Ad5 E1region (532-3525) as a Sall-BamH1 fragment into theSall-BamH1 cloning sites of the pHβApr-1-neo expression vector. Theserestriction sites were introduced by site-directed mutagenesis in theplasmid pXC38 which contains the Ad5 E1 region from nt 1-5788 (Xhol)subcloned in pBR322 between EcoRI and Sall (a generous gift of Dr. PhilBranton, McGill University, Montreal). The site-directed mutagenesis wasperformed lining the Transformer™ site-directed mutagenesis kit fromClonetech Laboratories Inc. (Gunning et al. 1987, Proc. Natl. Acad. Sci.USA 82:4831-4835). pHβE1AEIB (FIG. 1A) thus contains as a complementingelement the human Ad5 coding region spanning nucleotides 532 to 3525(Genebank M73260), which consists in the E1A gene, the E1B promotor, andthe E1B gene. The expression of E1A is not controlled by the natural E1Apromoter but by the strong constitutive human β-actin promotor. Asfurther shown in FIG. 1A pHβE1AE1B only harbors less than 200 bp overlapwith the parental adenoviral genome (3334-3525). Since it does notcontain the packaging and Ori sequences (the 5' ITR: 0-350), arecombination, although probabilistically infrequent, between theminimal overlap thereof and the Ad genome of the complementing elementin the E1complementing cell line should not give rise to a viableparticle, thereby eliminating the problem due to the presence of Ad E1+revert ants or RCA.

ENGINEERING OF THE BMAdE1 COMPLEMENTING CELL LINES

To construct the BMAdE1 complementing cell lines the A549 (human lungepithelial cells) were transfected with pHβE1AE1B (FIG.1A).

A total of 10 μg of purified pHβE1AE1, previously cut with ScaI, weretransfected onto A549 cells, a human lung carcinoma cell line (ATCC,CCL185) using the standard calcium phosphate precipitation technique. Itshould be understood that various methods of transfection arc well knownin the art and that the present invention is not limited to transfectionby the calcium phosphate procedure. For example, the vector could belipofected or electroplated.

INITIAL CHARACTERIZATION OF BMAdE1 CLONES

To determine whether or not the A549 cell lines expressed Ad E1A and E1Bfunctions, after transfection with pHβE1AE1B, they were infected withAdCMVlacZ tat a multiplicity of infection of 5-10 pfu/cell. Seventy-twohours later, cytopathy was apparent and the non-infected controls of thepositive cell lines were harvested, lysed, and analyzed on a Westernblot (FIG. 1B).

Results show that the expression of the E1B 19K and 55K proteins was notas high in the A549 cell lines as in 293 cells. However, in one of theA549 cell lines there was more E1A proteins than in 293 cells. It isconceivable that the cell lines which were less infected by AdCMVlacZexpressed less E1A and E1B. In other words that there is a correlationbetween the extent of infection and the expression of E1A and E1Bprotein from the complementing element.

The expression of adenovirus E1A and E1B products had already been shownin KB cells (Babiss et al., 1983 J. Virol. 46: 454-465). However the KBcomplementing cell lines of Babiss et al., are similar to 293 cellssince the whole E1region, including the 5' ITR and packaging sequencesare present in the complementing cell line and therefore does notdiminish the RCA problem encountered with 293 cells. In addition, theyields of infectious particles in these cells is generally lower than inthat of the reference complementing cell line, the 293 cell line.Consequently, the complementation exerted by the KB complementing celllines is only partial as compared to 293 cells. The present studiesdiffer from the previous work on two points. Firstly, the expression ofE1A products in the present construct is controlled by the strongerhuman beta-actin constitutive promotor, instead of the SV40 earlypromotor. This seems to ensure a better expression of the Ad E1proteins.Secondly, no cell lines were identified which only expressed E1Aproteins.

VIRAL PROGENY IN SELECTED BMAdE1CLONES

To assess the virus yield of the BMAdE1 clones, different clones wereinfected with AdCMVlacZ at a multiplicity of infection of 5-10 pfu/cell.The virus stock obtained with this infection was indirectly titered by abeta-gal chemiluminescent assay, the expression of lacZ being consideredproportional to the amount of infectious virus (Table 1).

                  TABLE 1    ______________________________________    Ad5CMVlacZ progeny comparison between BMAdE1    clones and 293 cells                 production    Ad5CMVlacZ   (PFU.sup.a per cell)                             in cell line:    ______________________________________    293          BMAdE1 78-42                             BMAdE1 220-8    800          200         800    ______________________________________     .sup.a Number of PFU was determined indirectly by gal expression in Hcla     S3 cells after 48 hours of infection with Ad5CMVlacZ stocks done on 293     and BMAdE1 clones.

The result show that the expression of lacZ, which reflects theexpression of the virus, in the BMAdE1 clones is delayed when comnparedto 293 cells (FIG. 2). Although the viral progeny of the BMAdE1 78-42clone is four times inferior to that of the 293 cells, the BMAdE1 220-8clone gives the same amount of infectious viral particules. Thus, inrelative terms, the BMAdE1 220-8 complementing cell line complements theE1-defect to the same extent as the 293 complementation cell line.

VIRAL TITRATION WITH THE SELECTED BMAdE1 CLONES

To determine the capacity of the BMAdE1 clones to plaque efficiently,dilutions or an AdGFP stock were plated on BMAdE1 78-42, BMAdE1 220-8,and 293 cells. The plaquing efficiency assessed the quality of theplaques and how easy it is to distinguish and count them following aproductive infection. The results are presented in Table 2.

                  TABLE 2    ______________________________________    Viral titers of a AdGFP stock.sup.a of BMAdE1    clones compared to 293 cells    AdGFP titer (PFU/ml)    in cell line:    ______________________________________    293         BMAdE1 78-42                            BMAdE1 220-8    7 × 10.sup.9                2 × 10.sup.9                            3 × 10.sup.9    ______________________________________     .sup.a AdGFP stock was made by infecting 5X10.sup.8 293 cells. Harvesting     was done at 48 hpi, and the pellet was resuspended in 50 ml of medium and     frozen/thawed three times.

Titers three and two fold inferior to that obtained with 293 cells wereobserved with the -78 and -220 clones, respectively.

For the AdGFP infection, the plaques were observed with an invertedfluorescence microscope, thereby allowing a visualization of at leasttwice the amount of plaques observable with the naked eye. This wasconfirmed by using bluo-gal™ for AdCMVlacZ infection.

The fact that BMAdE1 220-8 grows more in clusters as compared to BMAdE178-42 does not allow the easiest visualization of the plaques with thenaked eyes. Nevertheless, BMAdE1 clones can still be used toplaque-purify viruses after co-transfections, since one of the maingoals of the present invention is to get rid of the RCAs in virusstocks. In a preferred embodiment, 293 cells could be used for theinitial transfection. The scaling up of the production of infectiousE1-defective particles would be performed using the complementing celllines of the present invention, preferably BMAdE1 220, since it producesinfectious particles at the same level as 293 cells. The 293 cells couldalso be used for titering purposes in view of their good plaquingefficiency. A property which is somewhat shared by BMAdE1 78. When theRCA problem is not so acute, the 293S derivative, which grows insuspension, can be used for later stages of the preparation of theinfectious particles. The conventional 293 cell line as well as theBMAdE1 cell lines of the present invention all grow in monolayer andhence do not provide the ease of scaling up of cells growing insuspension. When both BMAdE1 and 293 cell lines are used for thepreparation of infectious particles it is preferable to use the latterfor the late passages, thereby avoiding the expansion of RCAs.

DEPOSITS

The E1-complementing cell lines BMAdE1-78-42 and BMAdE1 220-8 have beendeposited at the American Type Culture Collection (ATCC) 10801University Blvd., Manassas, Va. 20110-2209 under accession numbersCRL-12408 and CRL-12407, respectively.

CONCLUSION

In summary the present invention provides in particular,E1-complementing cell lines which overcome the problem of RCA productionand a recombinant vector for constructing such E1-complementing celllines. These cell lines avoid the emergence of E1+ revert ants duringmultiple passages and amplification of Ad helper-independent defectivevectors. It was herein demonstrated that the BMAdE1-220 cell lines cancomplement ΔE1Δ3Ad recombinants at the same level as 293 cells. Thiscell line allows the production of approximately 1000 infectiousparticles of Ad5CMVlacZ per cell, a value which is comparable to thatobtained in 293 cells (Table 1). This is in contrast to theE1-complementing cell lines obtained by Imler et al., (1996 Gene Ther.3:75-84), which "are able to support replication of E1-deletedadenoviruses, although not as efficiently as 293 cells (Table 1)".Indeed, the best complementing cell line obtained thereby yieldsapproximately 5 fold less infectious particles per cell, and in one caseas low as 100 fold less (Imler et al., 1996 Gene Ther. 3:75-84).

The BMAdE1-78 E1-complementing cell line while not being as good aproducer of infectious particles as BMAdE1-220 or 293 (producing about 4fold less per cell), provides however the advantage of showing a lesstransformed, rounded phenotype than BMAdE1-220, making it a better cellline for plaque purification.

Importantly, the cell lines of the present invention have not been shownto generate RCA during multiple passages. By providing a region ofhomology of less than 200 bp between the complementing cell lines of thepresent invention and the E1-defective adenoviral vectors, which is lessthan previously disclosed cell lines used, the likelyhood of RCAemergence is expected to be lower than that of previously disclosedcomplementing cell lines. In fact, no RCA is expected to emerge duringthe production of the stocks of infectious Ad particles using thecomplementing cell lines of the invention.

Finally, the expression of functional E1B proteins in the complementingcell lines of the present invention is thought to favor expression ofviral proteins and lead to superior yields of infectious virus particlesper cell. In addition E1B protein expression might diminish the knowntoxic effects that accompany E1A expression.

The present invention is not to be limited in scope by the recombinantconstructs and cell lines exemplified or deposited which are intended asbut single illustrations of one aspect of the invention. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying figures. Such modifications areintended to fall within the scope of the appended claims.

What is claimed is:
 1. An Ad E1-complementing cell line designatedBMAdE1-220-8, deposited with the ATCC under accession number CRL-12407.2. A method for large scale production of infectious E1-defectiveadenoviral particles:a) transfecting an E1-defective adenoviral vectorinto an E1-complementing cell line to obtain plaques; b) screening saidplaques to identify plaques which are positive for E1-defectiveadenovirus (Ad); c) submitting said E1-defective Ad of b) to at leasttwo rounds of plaque purification by infection into an E1-complementingcell line to obtain substantially pure infectious E1-defectiveadenoviral particles; and d) scaling up production of said substantiallypure infectious E1-defective adenoviral particles of c) by infecting anE1-complementing cell line and growing said cell line to obtain aconcentrated stock of infectious E1-defective adenoviral particles,wherein in at least one of steps a), b), c), and d), saidE1-complementing cell line is said Ad E1-complementing cell line ofclaim 1, thereby minimizing the production of replication competentadenovirus in said concentrated stock of infectious E1-defectiveadenoviral particles.